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標題: Expressing Infectious bursal disease virus vaccine by coupling Bamboo mosaic virus vector and plant transgenesis
作者: 袁琴雅
Chin-Ya Yuan
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摘要: 傳染性華氏囊病毒(Infectious bursal disease virus, IBDV)是一種急性且具高傳染性的病毒,為引起雞華氏囊病的主因,主要發生在 3-6 週的幼雛,常造成雞隻養殖場嚴重的經濟損失,因此疫苗的使用為當務之急。目前在此疫苗的發展上,除了一般的不活化病毒疫苗或減毒活疫苗之外,近幾年也有學者致力研究以植物做為生產平台,藉此以生產安全、方便且價格低廉之植物生產疫苗。因此,本研究分別使用 Bamboo mosaic virus (BaMV)、Tobacco mosaic virus(TMV)、Foxtail mosaic virus (FoMV)及 Potato virus X (PVX) 四種植物病毒做為載體,利用華氏囊病毒 VP2 基因取代病毒鞘蛋白,並於其 C 端融合 His-tag 形成功能缺失性的重組病 毒載體 BaMVdC-VP2His 、 TMVdC-VP2His 、FoMVdC-VP2His 及 PVXdC-VP2His,以農桿菌接種至菸草植物葉片上偵測 VP2His蛋白表現,結果顯示 4 種載體中以 BaMV 載體之 VP2His 蛋白表現量為最高。接著以菸草(Nicotiana benthamiana) 為材料 , 利用農桿菌轉殖方式建立BaMVdC-VP2His 轉殖植物,經西方墨點轉漬法篩選得到外表正常又能穩定表現蛋白的同源植物 (homologous line);由轉殖植物中純化 VP2 蛋白,於穿透式電子顯微鏡下可觀察到直徑大約為 20-30 nm 的 VP2 類病毒顆粒。為了顧及環境安全性和獲得更高的外源蛋白 以不同基因靜默抑制子(gene silencing suppressor),取代病毒三重疊基因區(triple gene block, TGB),利用基因靜默子和 BaMV 載體共同表現為策略,和嘗試共同表現竹嵌紋病毒鞘蛋白,結果顯示外源蛋白表現量並無明顯增加。為了研發食用疫苗,將 BaMVdC-VP2His 重組病毒基因體轉殖到大麥(Hordeum vulgare L.)中,雖然在大麥上的表現量不如預期,但已建立大麥轉殖的系統。未來由轉殖植物中純化出 VP2 類病毒顆粒進行動物試驗,偵測疫苗的中和抗性及效價,並以此系統發展次單位疫苗對抗雞華氏囊病毒。
Infectious bursal disease virus (IBDV) is an acute and highly contagious virus which is main cause of infectious bursal disease. It primarily occurrs in 3-6 weeks chickens and seriously affects the economy loss of poultry industry. Currently, inactivated and attenuated vaccines are two main strategies for chicken IBD prevention. In recent years, the safer and low-cost vaccines are produced by using plants as a platform. In this study, we constructed Bamboo mosaic virus (BaMV), Tobacco mosaic virus (TMV), Foxtail mosaic virus (FoMV), and Potato virus X (PVX) as plant viral vectors. The coat protein genes of viral vectors are replaced with IBDV VP2 gene and fused with His-tag in the C-terminal, to generate BaMVdC-VP2His, TMVdC-VP2His, FoMVdC-VP2His, and PVXdC-VP2His. To test the VP2 production, these four recombinant viruses were individually transfected into Nicotiana benthamiana leaves by agro-infiltration. The results showed that among four viral vectors, BaMV vector expressed the most high-level accumulation of VP2 protein. In order to stably express VP2 protein in plants, we generated BaMVdC-VP2His transgenic N. benthamiana plants by Agrobacterium- mediated transformation. Western blot analysis confirmed the VP2 protein could be expressed in homologous transgenic lines. The virus like particles (VLP) of VP2 approximately 20-30 nm in diameter were purified and observed by transmission electron microscopy. Taking into account the environmental safety and high expression of VP2His protein, different gene silencing suppressors replace BaMV TGB or co-expression with BaMVdC-VP2His. Furthermore, complementation coat protein function of BaMVdC-VP2His by co-expression of BaMV CP. The results indicate VP2 protein dose not significantly increase. To develop edible vaccine, the transgenic barley (Hordeum vulgare) were generated. The results showed that BaMVdC-VP2His transgenic barley accumulated low VP2 protein. However, we have successfully established tissue culture and plant regeneration system in barley. We will use VP2-VLP to immuning chicken and to test the neutralizing antibodies production. We hope in the future, to develop subunit vaccine against IBDV by the BaMV based vector system.
文章公開時間: 10000-01-01
Appears in Collections:生物科技學研究所



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